Pathogenesis and pathophysiology of inherited carnitine deficiency using carnitine-deficient JVS mice

1988年，金泽大学发现患有脂肪肝、高氨血症和低血糖的幼年型内脏脂肪变性（JVS）小鼠缺乏肉毒碱。我们描述了高氨血症是由抑制基因转录导致的尿素循环酶减少引起的。此外，我们发现JVS小鼠也表现出心肌肥大。在本研究中，我们重点研究了JVS小鼠的发病机制和病理生理。JVS小鼠的主要缺陷是肉碱在肾脏中的转运，这种转运是钠依赖的，对肉碱具有高亲和力，并被肉碱类似物、d -肉碱和γ -丁甜菜碱抑制。JVS小鼠尿液中排出大量的-丁甜菜碱。由于-丁甜菜碱是左旋肉碱生物合成的直接前体，而最后一步酶-丁甜菜碱-羟化酶在JVS小鼠中含量较高，因此我们认为左旋肉碱的生物合成途径是正常的，但由于前体在JVS小鼠中的重吸收缺陷而继发性缺陷。通过与Kanazawa小组的合作研究，我们在11号染色体上定位了jvs基因，位于微卫星位点D11Mit31附近。在JVS小鼠中，尿素循环酶基因的表达受到抑制，并伴有其他肝脏特异性酶或蛋白质基因如酪氨酸转氨酶和白蛋白的表达受到抑制。这些都是由糖皮质激素引起的。JVS小鼠血清糖皮质激素水平明显高于对照组，核区糖皮质激素受体蛋白丰富。另一方面，通过凝胶移位法检测到的核转录因子AP-1在JVS小鼠中非常活跃，并且在断奶期间变得活跃。根据这些结果，我们推断AP-1与糖皮质激素受体相互作用，从而抑制糖皮质激素对许多基因的诱导。为了将JVS小鼠肝脏中肉毒碱缺乏与AP-1激活联系起来，我们在原代培养的肝细胞中检测了脂肪酸和脂肪酸+肉毒碱对糖皮质激素诱导氨甲酰磷酸合成酶的影响。油酸、花生四酸等长链不饱和脂肪酸抑制糖皮质激素对CPS的诱导。添加肉毒碱后，脂肪酸对长链脂肪酸的抑制作用几乎完全消失，提示原代培养的肝细胞由于肉毒碱缺乏而不能代谢长链脂肪酸，模拟肉毒碱缺乏小鼠的情况。不饱和长链脂肪酸引起AP-1的活化。我们发现心脏肥厚的部分原因是通过激活儿茶酚胺代谢介导的。几种抑制剂对儿茶酚胺代谢的抑制引起心肌肥厚的部分抑制。为了更准确地阐明心肌肥厚的病理生理，我们对正常心室和肥厚心室mRNA表达进行了差异显示，发现了几种未知mRNA在心室之间表达不同。少

Carnitine is an essential component for the oxidation of fatty acid in mitochondria Juvenile visceral steatosis (JVS) mice suffering from fatty liver, hyperammonemia and hypoglycaemia, discovered at Kanazawa University in 1988, were found to be carnitine-deficient. We described that the hyperammonemia is caused by the decrease of the urea cycle enzymes which results from suppressed transcription of the genes. Furthermore, we found that JVS mice show cardiac hypertrophy, too. In this study, we focused our researches on the pathogenesis and pathophysiology of JVS mice. The primary defect of JVS mice was in the transport of carnitine in the kidney, which was sodium-dependent with a high affinity for carnitine and was inhibited by carnitine analogues, D-carnitine and gamma-butyrobetaine. Large amounts of gamma-butyrobetaine were excreted in the urine of JVS mice. Since gamma-butyrobetaine is the direct precursor of carnitine biosynthesis and since the last step enzyme, gamma-butyrobetaine … More hydroxylase, was rather higher in JVS mice, we conclude that the biosynthesis pathway of carnitine is normal but secondarily defective due to defective reabsorption of the precursor in JVS mice. By the cooperative study with Kanazawa group, we mapped the jvs gene on chromosome 11, in the vicinity of microsatellite locus D11Mit31.The suppressed expression of the urea cycle enzyme genes in JVS mice was accompanied with that of several other liver-specific enzyme or protein genes such as tyrosine aminotransferase and albumin. Those are all induced by glucocorticoid. Serum glucocorticoid levels of JVS mice were rather higher than tha controls and glucocorticoid receptor protein was abundant in the nuclear fraction. On the other hand, a nuclear transcription factor, AP-1, detected by gel-shift assay, was found to be very active in JVS mice and became activer during the weaning period. From these results, we reason that AP-1 interacts with glucocorticoid receptor and as the result, suppresses the induction by glucocorticoid of many genes. To connect the carnitine deficiency and AP-1 activation in the liver of JVS mice, we tested the effect of fatty acid and fatty acid+carnitine on the glucocorticoid induction of carbamylphosphate synthetase in the primary cultured hepatocytes. Long-chain unsaturated fatty acid, such as oleic acid and arachdonic acid, suppressed the induction of CPS by glucocorticoid. The suppression by fatty acids almost completely disappeared by the addition of carnitine, which suggests that the primary cultured hepatocytes can not metabolize long-chain fatty acid due to carnitine deficiency and mimic the situation of carnitine-deficient JVS mice. It seems that unsaturated long-chain fatty acids causes activation of AP-1. We found that some part of the cause of the cardiac hypertrophy was mediated by activation of catecholamine metabolism. The suppression of catecholamine metabolism by several inhibitors caused a partial suppression of cardiac hypertrophy. To clarify more precisely the pathophysiology of the cardiac hypertrophy, we adopted differential display for mRNAs expressed in normal and hypertrophied ventricles and found several unknown mRNA species expressed differently between the ventricles. Less